U.S. patent application number 16/637235 was filed with the patent office on 2020-07-23 for dna repair profiling and methods therefor.
The applicant listed for this patent is NANT HOLDINGS IP, LLC. Invention is credited to Kayvan NIAZI, Shahrooz RABIZADEH, Patrick SOON-SHIONG.
Application Number | 20200234790 16/637235 |
Document ID | / |
Family ID | 65271585 |
Filed Date | 2020-07-23 |
United States Patent
Application |
20200234790 |
Kind Code |
A1 |
SOON-SHIONG; Patrick ; et
al. |
July 23, 2020 |
DNA REPAIR PROFILING AND METHODS THEREFOR
Abstract
Systems and methods are contemplated that use various omics data
for DNA repair genes to assess a health associated parameter for an
individual.
Inventors: |
SOON-SHIONG; Patrick;
(Culver City, CA) ; RABIZADEH; Shahrooz; (Culver
City, CA) ; NIAZI; Kayvan; (Culver City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NANT HOLDINGS IP, LLC |
Culver City |
CA |
US |
|
|
Family ID: |
65271585 |
Appl. No.: |
16/637235 |
Filed: |
August 7, 2018 |
PCT Filed: |
August 7, 2018 |
PCT NO: |
PCT/US2018/045654 |
371 Date: |
February 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62542281 |
Aug 7, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G16H 50/30 20180101;
C12Q 1/6869 20130101; G16H 50/20 20180101; G16B 20/00 20190201;
G16H 20/10 20180101 |
International
Class: |
G16B 20/00 20060101
G16B020/00; C12Q 1/6869 20060101 C12Q001/6869; G16H 50/20 20060101
G16H050/20; G16H 50/30 20060101 G16H050/30; G16H 20/10 20060101
G16H020/10 |
Claims
1. A method of analyzing omics data, comprising: obtaining omics
data for a plurality of DNA damage repair genes, wherein the omics
data comprise at least two of DNA sequence data, RNA sequence data,
transcription strength, and protein activity or quantity; and
associating the omics data with at least one of a health status, an
omics error status, age, a disease, a prophylactic recommendation,
and a therapeutic recommendation.
2. The method of claim 1 further comprising a step of calculating a
score from the omics data to so obtain a health score.
3. The method of any one of the preceding claims wherein the DNA
sequence data are selected from the group consisting of mutation
data, copy number data duplication, loss of heterozygosity data,
and epigenetic status.
4. The method of any one of the preceding claims wherein the RNA
sequence data are selected from the group consisting of mRNA
sequence data and splice variant data.
5. The method of any one of the preceding claims wherein the RNA
sequence data are obtained from the group consisting of RNA from
solid tissue, RNA from blood cells, and circulating cell free
RNA.
6. The method of any one of the preceding claims wherein the
transcription strength is expressed as transcripts of the damage
repair gene per million transcripts.
7. The method of any one of the preceding claims wherein the
protein activity or quantity is determined using a mass
spectroscopic method.
8. The method of any one of the preceding claims wherein the health
status is selected from the group consisting of healthy, diagnosed
with an age related disease, and diagnosed with cancer.
9. The method of any one of the preceding claims wherein the
prophylactic recommendation comprises a recommendation to treat an
individual with an agent that modulates expression of at least one
of the plurality of DNA damage repair genes.
10. The method of any one of the preceding claims wherein the
therapeutic recommendation comprises a recommendation to treat a
patient with a DNA damaging agent.
11. The method of any one of the preceding claims wherein the
plurality of DNA damage repair genes is selected from at least one
of a base excision repair gene, a mismatch repair gene, a
nucleotide excision repair gene, a homologous recombination gene,
and a non-homologous end-joining gene.
12. The method of any one of the preceding claims wherein the
plurality of DNA damage repair genes is selected from at least two
of a base excision repair gene, a mismatch repair gene, a
nucleotide excision repair gene, a homologous recombination gene,
and a non-homologous end-joining gene.
13. The method of any one of the preceding claims wherein the
plurality of DNA damage repair genes is selected from at least
three of a base excision repair gene, a mismatch repair gene, a
nucleotide excision repair gene, a homologous recombination gene,
and a non-homologous end-joining gene.
14. The method of any one of the preceding claims wherein the
plurality of DNA damage repair genes is selected from a base
excision repair gene, a mismatch repair gene, a nucleotide excision
repair gene, a homologous recombination gene, and a non-homologous
end-joining gene.
15. The method of any one of the preceding claims wherein the
plurality of DNA damage repair genes are at least two genes
selected from the genes listed in Table 1, Table 2, and Table
3.
16. The method of any one of the preceding claims wherein the step
of associating comprises a weight score for at least one of the
omics data.
17. The method of any one of the preceding claims further
comprising a step of comparing the omics error status with a
threshold value to thereby determine a risk score. (tipping
point')
18. A method of calculating a health indicator, comprising:
obtaining omics data for a plurality of DNA damage repair genes,
wherein the omics data comprise at least two of DNA sequence data,
RNA sequence data, transcription strength, and protein activity or
quantity; and using the omics data for the plurality of DNA damage
repair genes to generate a health compound score that is indicative
of the health of a person.
19. The method of claim 18 further comprising a step of comparing
the compound score with a threshold value to thereby determine a
treatment option.
20. The method of claim 19 wherein the treatment option is a
prophylactic treatment where the compound score is below the
threshold value.
21. The method of claim 19 wherein the treatment option uses a drug
that modulates expression of at least one of the plurality of DNA
damage repair genes.
22. The method of claim 19 wherein the treatment option uses a drug
that induces DNA damage.
23. A method of treating an individual, comprising: obtaining omics
data for a plurality of DNA damage repair genes, wherein the omics
data comprise at least two of DNA sequence data, RNA sequence data,
transcription strength, and protein activity or quantity;
identifying at least one of the DNA damage repair genes as being
dysregulated relative to a corresponding healthy control; and
administering an agent that counteracts the at least one of the
dysregulated DNA damage repair gene.
24. The method of claim 23 wherein the DNA sequence data are
selected from the group consisting of mutation data, copy number
data duplication, loss of heterozygosity data, and epigenetic
status.
25. The method of any one of claims 23-24 wherein the RNA sequence
data are selected from the group consisting of mRNA sequence data
and splice variant data.
26. The method of any one of claims 23-25 wherein the RNA sequence
data are obtained from the group consisting of RNA from solid
tissue, RNA from blood cells, and circulating cell free RNA.
27. The method of any one of claims 23-26 wherein the transcription
strength is expressed as transcripts of the damage repair gene per
million transcripts.
28. The method of any one of claims 23-27 wherein the protein
activity or quantity is determined using a mass spectroscopic
method.
29. The method of any one of claims 23-28 wherein the at least one
of the DNA damage repair gene is selected from a base excision
repair gene, a mismatch repair gene, a nucleotide excision repair
gene, a homologous recombination gene, and a non-homologous
end-joining gene.
30. The method of any one of claims 23-28 wherein the plurality of
DNA damage repair genes are at least two genes selected from the
genes listed in Table 1, Table 2, and Table 3.
31. A method of performing a test on a subject, comprising:
obtaining a blood sample from the subject; using the blood sample
to obtain omics data for a plurality of DNA damage repair genes,
wherein the omics data comprise at least two of DNA sequence data,
RNA sequence data, transcription strength, and protein activity or
quantity; wherein the omics data are obtained from at least one of
a cell free portion of the blood sample and a cell containing
portion of the blood sample; identifying at least one of the DNA
damage repair genes in the blood sample as being dysregulated
relative to a corresponding healthy control.
32. The method of claim 31 wherein the omics data are obtained from
the cell free portion of the blood sample.
33. The method of any one of claims 31-32 wherein the RNA sequence
data are selected from the group consisting of mRNA sequence data
and splice variant data.
34. The method of any one of claims 31-33 wherein the RNA sequence
data are obtained from the group consisting of RNA from solid
tissue, RNA from blood cells, and circulating cell free RNA.
35. The method of any one of claims 31-34 wherein the transcription
strength is expressed as transcripts of the damage repair gene per
million transcripts.
36. The method of any one of claims 31-35 wherein the at least one
of the DNA damage repair gene is selected from a base excision
repair gene, a mismatch repair gene, a nucleotide excision repair
gene, a homologous recombination gene, and a non-homologous
end-joining gene.
37. The method of any one of claims 31-35 wherein the plurality of
DNA damage repair genes are at least two genes selected from the
genes listed in Table 1, Table 2, and Table 3.
Description
[0001] This application claims priority to our copending U.S.
provisional application with the Ser. No. 62/542,281, which was
filed Aug. 7, 2017.
FIELD OF THE INVENTION
[0002] The field of the invention is profiling of omics data as
they relate to DNA repair, and especially as it relates to the
generation of a global health indicator, and to prophylactic and
therapeutic methods and compositions to counteract age-related
conditions and diseases.
BACKGROUND OF THE INVENTION
[0003] The following description includes information that may be
useful in understanding the present invention. It is not an
admission that any of the information provided herein is prior art
or relevant to the presently claimed invention, or that any
publication specifically or implicitly referenced is prior art.
[0004] In addition to the inherent error-prone nature of various
DNA polymerases, mammalian DNA is constantly subjected to chemical,
physical, and metabolic challenges that can introduce chemical
changes, loss of nucleobases, and DNA single and double strand
breaks. Indeed, it is estimated that each of the approximately
10.sup.13 cells within the human body incurs tens of thousands of
DNA-damaging events per day (see e.g., Lindahl T, Barnes DE (2000)
Repair of endogenous DNA damage. Cold Spring Harb Symp Quant Biol
65:127-133). All publications and patent applications identified
herein are incorporated by reference to the same extent as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Where a
definition or use of a term in an incorporated reference is
inconsistent or contrary to the definition of that term provided
herein, the definition of that term provided herein applies and the
definition of that term in the reference does not apply.
[0005] While such damage often results in genomic instability and
cell death, many of these lesions also cause structural damage to
DNA and can alter or eliminate fundamental cellular processes, such
as DNA replication or transcription. To counteract the harmful
effects of DNA damage, cells have various DNA repair systems,
including base excision repair, mismatch repair, nucleotide
excision repair, and double-strand break repair, which comprise
both homologous recombination and non-homologous end-joining.
[0006] Previous experimental data on animals having defects in DNA
repair genes often showed a decreased life span and increased
cancer incidence. For example, mice that were deficient in the
dominant NHEJ (non-homologous end-joining) pathway and in telomere
maintenance mechanisms were prone to lymphoma and infections, and
typically had shorter lifespans than wild-type mice. In a similar
manner, mice that were deficient in a key repair and transcription
protein that unwinds DNA helices had often premature onset of
age-related diseases and shortening of lifespan. However, the
effects of deficiencies in DNA repair are not readily predictable:
mice having a deficient NER pathway tend to exhibit shortened life
span without correspondingly higher rates of mutation. With further
respect to cancer, various known DNA repair gene mutations are
associated with increased cancer risk. For example, hereditary
nonpolyposis colorectal cancer (HNPCC) is strongly associated with
specific mutations in the DNA mismatch repair pathway, while BRCA1
and BRCA2 are associated in breast cancer with a large number of
DNA repair pathways, especially NHEJ and homologous recombination.
More recently, mutations in DNA repair genes were also implicated
in cancer metastases (see e.g., Radiation Research 181, 111-130
(2014)). However, no discernible pattern exists for DNA repair
genes that could be used to predict the effect of an increased or
decreased activity of a particular DNA repair pathway.
[0007] Therefore, while numerous experimental details are known for
DNA repair genes and pathways, there is a lack of systemic
understanding and use of DNA repair genes and pathways in the
assessment of health and treatment recommendations.
SUMMARY OF THE INVENTION
[0008] The inventive subject matter provides systems and methods in
which multiple omics data for various DNA repair genes of a patient
sample are employed to derive one or more health associated
parameter. For example, preferred omics data include DNA sequence
data, RNA sequence data, and particularly transcription strength,
and/or protein activity or protein quantity, while especially
preferred health associated parameters include health status, error
status, and treatment recommendations. Moreover, expression levels
(transcription strength) of various DNA repair genes can be used to
assess real-time status of the DNA repair system to indicate
overall health, presence and/or severity of DNA damage (due to
environmental factors or pharmaceutical intervention), and as such
can be used to monitor response to a treatment or to predict
recurrence of disease.
[0009] In one aspect of the inventive subject matter, the inventors
contemplate a method of analyzing omics data that includes a step
of obtaining omics data for a plurality of DNA damage repair genes,
wherein the omics data comprise at least two of DNA sequence data,
RNA sequence data, transcription strength, and protein activity or
quantity. In another step, the omics data are then associated with
a health status, an omics error status, age, a disease, a
prophylactic recommendation, and/or a therapeutic recommendation.
Where desired, contemplated methods may further include a step of
calculating a score from the omics data to so obtain a health
score.
[0010] With respect to DNA sequence data it is contemplated that
such data may include mutation data, copy number data duplication,
loss of heterozygosity data, and/or epigenetic status, while RNA
sequence data may include mRNA sequence data and splice variant
data, which may be obtained from solid tissue, from blood cells,
and/or from circulating cell free RNA. Moreover, it is generally
preferred that the transcription strength is expressed as
transcripts of the damage repair gene per million transcripts,
and/or that protein activity or quantity is determined using a mass
spectroscopic method (e.g., using a selective reaction monitoring
method).
[0011] The health status may typically include a healthy status, a
diagnosis with an age related disease, and a diagnosis with cancer.
Contemplated prophylactic recommendation will include a
recommendation to treat an individual with an agent that modulates
expression of at least one of the plurality of DNA damage repair
genes, while therapeutic recommendations may comprise a
recommendation to treat a patient with a DNA damaging agent.
Suitable DNA damage repair genes will include one or more of a base
excision repair gene, a mismatch repair gene, a nucleotide excision
repair gene, a homologous recombination gene, and a non-homologous
end-joining gene, and exemplary DNA damage repair genes are listed
in Tables 1-3 below.
[0012] Contemplated steps of associating the omics data with a
status may comprise a weight score for at least one of the omics
data, and it is further contemplated that such method may further
comprise a step of comparing the omics error status with a
threshold value to thereby determine a risk score.
[0013] Therefore, and viewed from a different perspective, the
inventors also contemplate a method of calculating a health
indicator that includes a step of obtaining omics data for a
plurality of DNA damage repair genes, wherein the omics data
comprise at least two of DNA sequence data, RNA sequence data,
transcription strength, and protein activity or quantity. The so
determined omics data are then used to generate a health compound
score that is indicative of the health of a person.
[0014] As noted above, contemplated methods may further comprise a
step of comparing the compound score with a threshold value to
thereby determine a treatment option. For example, the treatment
option may be a prophylactic treatment where the compound score is
below the threshold value, the treatment option may use a drug that
modulates expression of at least one of the plurality of DNA damage
repair genes, or the treatment option may use a drug that induces
DNA damage.
[0015] In yet another aspect of the inventive subject matter, the
inventors also contemplate a method of treating an individual that
includes the steps of obtaining omics data for a plurality of DNA
damage repair genes, wherein the omics data comprise at least two
of DNA sequence data, RNA sequence data, transcription strength,
and protein activity or quantity, and a further step of identifying
at least one of the DNA damage repair genes as being dysregulated
relative to a corresponding healthy control. In yet another step,
an agent is then administered that counteracts the at least one of
the dysregulated DNA damage repair gene.
[0016] Most typically, DNA sequence data are selected from the
group consisting of mutation data, copy number data duplication,
loss of heterozygosity data, and epigenetic status, while the RNA
sequence data are selected from the group consisting of mRNA
sequence data and splice variant data. As noted the RNA sequence
data may be obtained from solid tissue, blood cells, and/or
circulating cell free RNA. Most typically, the transcription
strength is expressed as transcripts of the damage repair gene per
million transcripts, and/or the protein activity or quantity is
determined using a mass spectroscopic method. With respect to the
DNA damage repair genes it is contemplated that the at least one or
more of the DNA damage repair genes a base excision repair gene, a
mismatch repair gene, a nucleotide excision repair gene, a
homologous recombination gene, and/or a non-homologous end-joining
gene. For example, suitable DNA damage repair genes are listed in
Table 1, Table 2, and Table 3.
[0017] Therefore, the inventors also contemplate a method of
performing a test on a subject that includes a step of obtaining a
blood sample from the subject, and another step of using the blood
sample to obtain omics data for a plurality of DNA damage repair
genes, wherein the omics data comprise at least two of DNA sequence
data, RNA sequence data, transcription strength, and protein
activity or quantity. Most preferably, the omics data are obtained
from a cell free portion of the blood sample and/or a cell
containing portion of the blood sample. In still another step of
contemplated methods, at least one of the DNA damage repair genes
is identified in the blood sample as being dysregulated relative to
a corresponding healthy control.
[0018] Most typically, the RNA sequence data are selected from the
group consisting of mRNA sequence data and splice variant data, and
the RNA sequence data may be obtained from solid tissue, from blood
cells, and/or circulating cell free RNA. The transcription strength
is preferably expressed as transcripts of the damage repair gene
per million transcripts. As noted above, preferred DNA damage
repair genes are selected from a base excision repair gene, a
mismatch repair gene, a nucleotide excision repair gene, a
homologous recombination gene, and a non-homologous end-joining
gene. For example, exemplary DNA damage repair genes include those
listed in Table 1, Table 2, and Table 3.
[0019] Various objects, features, aspects and advantages of the
inventive subject matter will become more apparent from the
following detailed description of preferred embodiments, along with
the accompanying drawing figures in which like numerals represent
like components.
BRIEF DESCRIPTION OF THE DRAWING
[0020] FIG. 1 is an exemplary illustration of various modes of DNA
damage, resultant lesions, and repair pathways to counteract such
damage.
DETAILED DESCRIPTION
[0021] The inventors have now discovered that a library or
reference database for all DNA repair genes can be created using
one or more omics data for each gene associated with DNA repair,
and that such library is particularly useful where the omics data
are associated with one or more health parameter. Such library or
reference database may be particularly useful where expression
levels of DNA damage repair genes are quantified and/or where
mutations (and particularly mutations affecting DNA repair) are
detected, and where such quantities and detected mutations are
associated with a particular health status.
[0022] Viewed from a different perspective, the inventors
contemplate that signatures for omics data from DNA repair
associated genes can be identified that are characteristic for the
error status within a patient, which in turn may be indicative for
one or more health related conditions. Likewise, such signatures
may be predictive of DNA damage even before the actual damage can
be observed in a diseased tissue. As will be readily appreciated,
signatures may be ascertained once (e.g., during a routine visit
before signs or symptoms of a disease are evidence), or be followed
over time for a single patient, which may be especially useful
where health is generally assessed, or where a disease or treatment
is monitored.
[0023] While traditional studies of DNA repair have often focused
on the presence or strength of expression of a particular gene
associated within a single DNA repair pathway, the inventors now
contemplate that such analysis is insufficient to obtain an
indicator that has predictive or even analytic power with respect
to a health condition or likely treatment outcome. To that end, the
inventors have discovered that DNA repair genes can be analyzed not
only as present or absent, but that a full scale omics analysis
will take into account multiple aspects of multiple genes. More
specifically, the inventors contemplate a library or reference
database that catalogs not only DNA sequence data of DNA repair
associated genes, but also corresponding RNA sequence data,
corresponding transcription strength, and corresponding protein
activity and/or quantity of multiple DNA repair associated genes to
so provide a dynamic picture of DNA repair activity.
[0024] Advantageously, and particularly where contemplated
signatures for DNA repair associated genes (e.g., expression levels
of one or more DNA repair associated genes) are combined with omics
data from diseased tissue, mutational patterns in diseased tissue
can be correlated with the signatures for confirmation of treatment
as well as prediction of treatment outcome. Moreover, where
contemplated signatures for DNA repair associated genes include
analyses for gene damage in the DNA repair associated genes, such
mutational damage may be predictive for hypermutations in the tumor
genome due to lack of an efficient repair system.
[0025] Therefore, DNA sequence data will not only include the
presence or absence of a gene that is associated with DNA repair,
but also take into account mutation data where the repair
associated gene is mutated, the copy number (e.g., to identify
duplication, loss of allele or heterozygosity), and even epigenetic
status (e.g., methylation, histone phosphorylation, nucleosome
positioning, etc.). With respect to RNA sequence data it should be
noted that contemplated RNA sequence data include mRNA sequence
data, splice variant data, polyadenylation information, etc.
Moreover, it is generally preferred that the RNA sequence data also
include a metric for the transcription strength (e.g., number of
transcripts of a damage repair gene per million total transcripts,
number of transcripts of a damage repair gene per number of
transcripts for actin or other household gene RNA, etc.). It should
be noted that such transcription strength information is
particularly useful where transcription strength is measured over
time to detect an increase in a particular type of DNA damage.
Similarly, it is generally preferred that contemplated analyses may
also include one or more metrics that can quantify protein activity
and/or protein quantity for a particular gene associated with DNA
repair. For example, suitable protein activity or quantity can be
determined using known enzymatic assays, and/or various mass
spectroscopic methods, and especially selected reaction monitoring
methods such as multiple reaction monitoring and parallel reaction
monitoring.
[0026] Of course, it should be noted that the omics data can be
obtained in numerous manners and from numerous sources, and
especially preferred source materials include whole blood and
cell-containing and cell-free portions thereof, and tissue biopsies
from diseased and/or healthy organs of an individual. For example,
DNA and RNA may be obtained from solid tissue, from blood cells,
and/or from a pool of circulating cell free RNA. In other examples,
DNA, RNA, and/or protein may be obtained from a tissue biopsy
(e.g., fresh, frozen, or FFPE), which may be collected together
with a sample of corresponding healthy tissue. In further preferred
aspects, omics data can also be obtained from single cell
sequencing. Moreover and as already noted earlier, the omics data
can be obtained from more than one tissue or source, and over
multiple points in time. For example, omics data may be initially
obtained from biopsy material of a diseased tissue and a further
non-diseased sample of the same patient (e.g., skin, blood, etc.).
Alternatively, or additionally, omics data may be initially
obtained from circulating nucleic acids (and especially cfRNA
(circulating cell free RNA)) of a blood draw or other biological
fluid, alone or in combination with omics data from healthy and/or
diseased tissue.
[0027] Advantageously, it should be noted that the omics data can
also be obtained at a point in time prior to a treatment (or even a
diagnosis), during treatment, and/or after a treatment. Similarly,
omics data can be obtained prior to or after exposure to a
particular environment (e.g., prior to entry into a chemically or
radiologically contaminated area), or prior to or after exposure to
a particular DNA damaging condition (e.g., sun exposure, RF
exposure, etc.). As will be readily appreciated, repeated
acquisition of omics data will allow identification of trends in
triggering or maintenance of a DNA damage response, which in turn
specifically indicates the type and severity of cellular
stress.
[0028] In addition, it is contemplated that the omics data for the
genes associated with DNA repair may be acquired in parallel (or at
some other time) with omics data for non-repair relevant genes that
are specific for a diseased tissue. For example, conventional
nucleic acid analysis will typically only identify mutations of a
tumor tissue relative to normal tissue. In contrast, contemplated
analyses may include omics data for genes associated with DNA
repair together with omics data of non-repair relevant genes
specific for a diseased tissue (tumor specific mutations or tumor
specific changes in gene expression). Such analysis is especially
useful where the omics data for the non-repair relevant genes are
used in pathway analysis as such combined data will not only allow
identification of activity and status of genes associated with DNA
repair, but also physiological activity that is relevant in the
context of DNA repair. For example, pathway analysis may reveal
that certain pathways (e.g., apoptosis or other cell death relevant
pathway) are activated where activity of genes associated with DNA
repair is increased, which may be indicative of a treatment
success. On the other hand, other pathways may be activated (e.g.,
pathways associated with EMT) where activity of genes associated
with DNA repair is increased, which may be indicative of potential
treatment failure. Therefore, contemplated combined analyses will
add further functional information of a cell in the context of cell
stress and DNA repair.
[0029] As should also be readily appreciated, the type of omics
data will vary considerably and will typically depend on the type
of sample used, omics parameter (e.g., genomic data, transcriptomic
data, proteomic data, etc.), and/or desired omics data
characteristic (e.g., mutational information, strength of
transcription, protein activity, pathway activity, etc.).
Consequently, suitable omics data include as raw data (e.g.,
FASTQ), differential data (e.g., after BAMBAM analysis), various
processed data (e.g., VCF format), or even as data after analysis
using pathway analysis (e.g., using PARADIGM).
[0030] Therefore, and viewed from a different perspective, it
should be appreciated that omics analysis across multiple genes
associated with DNA repair the library will provide a detailed
insight with respect to integrity and/or activity of DNA repair
associated genes and pathways, and as such allows for a
quantitative analysis of the overall mutation status of a genome,
and more particularly of the mutation and functional status of the
DNA repair mechanisms in a patient or other individual.
[0031] With respect to contemplated genes associated with DNA
repair, Table 1 provides an exemplary collection of predominant DNA
repair genes and their associated repair pathways presented herein,
and a typical library of genes associated with DNA repair will
include one, or two, or three, or four, or more of at least two
repair categories of Table 1.
TABLE-US-00001 TABLE 1 Repair mechanism Predominant DNA Repair
genes Base excision repair (BER) DNA glycosylase, APE1, XRCC1,
PNKP, Tdp1, APTX, DNA polymerase .beta., FEN1, DNA polymerase
.delta. or .epsilon., PCNA-RFC, PARP Mismatch repair (MMR)
MutS.alpha. (MSH2-MSH6), MutS.beta. (MSH2-MSH3), MutL.alpha. (MLH1-
PMS2), MutL.beta. (MLH1-PMS2), MutL.gamma. (MLH1-MLH3), Exo1,
PCNA-RFC XPC-Rad23B-CEN2, UV-DDB (DDB1-XPE), CSA, CSB, TFIIH,
Nucleotide excision repair (NER) XPB, XPD, XPA, RPA, XPG,
ERCC1-XPF, DNA polymerase .delta. or .epsilon. Homologous
recombination (HR) Mre11-Rad50-Nbs1, CtIP, RPA, Rad51, Rad52,
BRCA1, BRCA2, Exo1, BLM-TopIII.alpha., GEN1-Yen1, Slx1-Slx4,
Mus81/Eme1 Non-homologous end-joining Ku70-Ku80, DNA-PKc, XRCC4-DNA
ligase IV, XLF (NHEJ)
[0032] However, it should be recognized that numerous other genes
associated with DNA repair and repair pathways are also expressly
contemplated herein, and Tables 2 and 3 illustrate further
exemplary genes for analysis and their associated function in DNA
repair.
TABLE-US-00002 TABLE 2 Accession Gene name (synonyms) Activity
number Base excision repair (BER) DNA glycosylases: major altered
base released UNG U excision NM_003362 SMUG1 U excision NM_014311
MBD4 U or T opposite G at CpG sequences NM_003925 TDG U, T or
ethenoC opposite G NM_003211 OGG1 8-oxoG opposite C NM_002542 MYH A
opposite 8-oxoG NM_012222 NTH1 Ring-saturated or fragmented
pyrimidines NM_002528 MPG 3-meA, ethenoA, hypoxanthine NM_002434
Other BER factors APE1 (HAP1, APEX, REF1) AP endonuclease NM_001641
APE2 (APEXL2) AP endonuclease NM_014481 LIG3 Main ligation function
NM_013975 XRCC1 Main ligation function NM_006297 Poly(ADP-ribose)
polymerase (PARP) enzymes ADPRT Protects strand interruptions
NM_001618 ADPRTL2 PARP-like enzyme NM_005485 ADPRTL3 PARP-like
enzyme AF085734 Direct reversal of damage MGMT O6-meG
alkyltransferase NM_002412 Mismatch excision repair (MMR) MSH2
Mismatch and loop recognition NM_000251 MSH3 Mismatch and loop
recognition NM_002439 MSH6 Mismatch recognition NM_000179 MSH4 MutS
homolog specialized for meiosis NM_002440 MSH5 MutS homolog
specialized for meiosis NM_002441 PMS1 Mitochondrial MutL homolog
NM_000534 MLH1 MutL homolog NM_000249 PMS2 MutL homolog NM_000535
MLH3 MutL homolog of unknown function NM_014381 PMS2L3 MutL homolog
of unknown function D38437 PMS2L4 MutL homolog of unknown function
D38438 Nucleotide excision repair (NER) XPC Binds damaged DNA as
complex NM_004628 RAD23B (HR23B) Binds damaged DNA as complex
NM_002874 CETN2 Binds damaged DNA as complex NM_004344 RAD23A
(HR23A) Substitutes for HR23B NM_005053 XPA Binds damaged DNA in
preincision NM_000380 complex RPA1 Binds DNA in preincision complex
NM_002945 RPA2 Binds DNA in preincision complex NM_002946 RPA3
Binds DNA in preincision complex NM_002947 TFIIH Catalyzes
unwinding in preincision complex XPB (ERCC3) 3' to 5' DNA helicase
NM_000122 XPD (ERCC2) 5' to 3' DNA helicase X52221 GTF2H1 Core
TFIIH subunit p62 NM_005316 GTF2H2 Core TFIIH subunit p44 NM_001515
GTF2H3 Core TFIIH subunit p34 NM_001516 GTF2H4 Core TFIIH subunit
p52 NM_001517 CDK7 Kinase subunit of TFIIH NM_001799 CCNH Kinase
subunit of TFIIH NM_001239 MNAT1 Kinase subunit of TFIIH NM_002431
XPG (ERCC5) 3' incision NM_000123 ERCC1 5' incision subunit
NM_001983 XPF (ERCC4) 5' incision subunit NM_005236 LIG1 DNA
joining NM_000234 NER-related CSA (CKN1) Cockayne syndrome; needed
for NM_000082 transcription-coupled NER CSB (ERCC6) Cockayne
syndrome; needed for NM_000124 transcription-coupled NER XAB2
(HCNP) Cockayne syndrome; needed for NM_020196
transcription-coupled NER DDB1 Complex defective in XP group E
NM_001923 DDB2 Mutated in XP group E NM_000107 MMS19 Transcription
and NER AW852889 Homologous recombination RAD51 Homologous pairing
NM_002875 RAD51L1 (RAD51B) Rad51 homolog U84138 RAD51C Rad51
homolog NM_002876 RAD51L3 (RAD51D) Rad51 homolog NM_002878 DMC1
Rad51 homolog, meiosis NM_007068 XRCC2 DNA break and cross-link
repair NM_005431 XRCC3 DNA break and cross-link repair NM_005432
RAD52 Accessory factor for recombination NM_002879 RAD54L Accessory
factor for recombination NM_003579 RAD54B Accessory factor for
recombination NM_012415 BRCA1 Accessory factor for transcription
and NM_007295 recombination BRCA2 Cooperation with RAD51, essential
NM_000059 function RAD50 ATPase in complex with MRE11A, NBS1
NM_005732 MRE11A 3' exonuclease NM_005590 NBS1 Mutated in Nijmegen
breakage syndrome NM_002485 Nonhomologous end-joining Ku70 (G22P1)
DNA end binding NM_001469 Ku80 (XRCC5) DNA end binding M30938 PRKDC
DNA-dependent protein kinase catalytic NM_006904 subunit LIG4
Nonhomologous end-joining NM_002312 XRCC4 Nonhomologous end-joining
NM_003401 Sanitization of nucleotide pools MTH1 (NUDT1) 8-oxoGTPase
NM_002452 DUT dUTPase NM_001948 DNA polymerases (catalytic
subunits) POLB BER in nuclear DNA NM_002690 POLG BER in
mitochondrial DNA NM_002693 POLD1 NER and MMR NM_002691 POLE1 NER
and MMR NM_006231 PCNA Sliding clamp for pol delta and pol epsilon
NM_002592 REV3L (POLZ) DNA pol zeta catalytic subunit, essential
NM_002912 function REV7 (MAD2L2) DNA pol zeta subunit NM_006341
REV1 dCMP transferase NM_016316 POLH XP variant NM_006502 POLI
(RAD30B) Lesion bypass NM_007195 POLQ DNA cross-link repair
NM_006596 DINB1 (POLK) Lesion bypass NM_016218 POLL Meiotic
function NM_013274 POLM Presumed specialized lymphoid function
NM_013284 TRF4-1 Sister-chromatid cohesion AF089896 TRF4-2
Sister-chromatid cohesion AF089897 Editing and processing nucleases
FEN1 (DNase IV) 5' nuclease NM_004111 TREX1 (DNase III) 3'
exonuclease NM_007248 TREX2 3' exonuclease NM_007205 EX01 (HEX1) 5'
exonuclease NM_003686 SPO11 endonuclease NM_012444 Rad6 pathway
UBE2A (RAD6A) Ubiquitin-conjugating enzyme NM_003336 UBE2B (RAD6B)
Ubiquitin-conjugating enzyme NM_003337 RAD18 Assists repair or
replication of damaged AB035274 DNA UBE2VE (MMS2)
Ubiquitin-conjugating complex AF049140 UBE2N (UBC13, BTG1)
Ubiquitin-conjugating complex NM_003348 Genes defective in diseases
associated with sensitivity to DNA damaging agents BLM Bloom
syndrome helicase NM_000057 WRN Werner syndrome
helicase/3'-exonuclease NM_000553 RECQL4 Rothmund-Thompson syndrome
NM_004260 ATM Ataxia telangiectasia NM_000051 Fanconi anemia FANCA
Involved in tolerance or repair of DNA NM_000135 cross-links FANCB
Involved in tolerance or repair of DNA N/A cross-links FANCC
Involved in tolerance or repair of DNA NM_000136 cross-links FANCD
Involved in tolerance or repair of DNA N/A cross-links FANCE
Involved in tolerance or repair of DNA NM_021922 cross-links FANCF
Involved in tolerance or repair of DNA AF181994 cross-links FANCG
(XRCC9) Involved in tolerance or repair of DNA NM_004629
cross-links Other identified genes with a suspected DNA repair
function SNM1 (PS02) DNA cross-link repair D42045 SNM1B Related to
SNM1 AL137856 SNM1C Related to SNM1 AA315885 RPA4 Similar to RPA2
NM_013347 ABH (ALKB) Resistance to alkylation damage X91992 PNKP
Converts some DNA breaks to ligatable NM_007254 ends Other
conserved DNA damage response genes ATR ATM- and PI-3K-like
essential kinase NM_001184 RAD1 (S. pombe) homolog PCNA-like DNA
damage sensor NM_002853 RAD9 (S. pombe) homolog PCNA-like DNA
damage sensor NM_004584 HUS1 (S. pombe) homolog PCNA-like DNA
damage sensor NM_004507 RAD17 (RAD24) RFC-like DNA damage sensor
NM_002873 TP53BP1 BRCT protein NM_005657 CHEK1 Effector kinase
NM_001274 CHK2 (Rad53) Effector kinase NM_007194
TABLE-US-00003 TABLE 3 Gene Name Gene Title Biological Activity
RFC2 replication factor C (activator 1) 2, DNA replication 40 kDa
XRCC6 X-ray repair complementing defective DNA ligation///DNA
repair///double-strand break repair in Chinese hamster cells 6 (Ku
repair via nonhomologous end-joining///DNA autoantigen, 70 kDa)
recombination///positive regulation of transcription,
DNA-dependent///double-strand break repair via nonhomologous
end-joining///response to DNA damage stimulus///DNA recombination
APOBEC apolipoprotein B mRNA editing enzyme, For all of APOBEC1,
APOBEC2, APOBEC3A-H, and catalytic polypeptide-like APOBEC4,
cytidine deaminases. POLD2 polymerase (DNA directed), delta 2, DNA
replication///DNA replication regulatory subunit 50 kDa PCNA
proliferating cell nuclear antigen regulation of progression
through cell cycle///DNA replication///regulation of DNA
replication///DNA repair///cell
proliferation///phosphoinositide-mediated signaling///DNA
replication RPA1 replication protein A1, 70 kDa DNA-dependent DNA
replication///DNA repair///DNA recombination///DNA replication RPA1
replication protein A1, 70 kDa DNA-dependent DNA replication///DNA
repair///DNA recombination///DNA replication RPA2 replication
protein A2, 32 kDa DNA replication///DNA-dependent DNA replication
ERCC3 excision repair cross-complementing DNA topological
change///transcription-coupled rodent repair deficiency,
nucleotide-excision repair///transcription///regulation
complementation group 3 (xeroderma of transcription,
DNA-dependent///transcription from pigmentosum group B
complementing) RNA polymerase II promoter///induction of
apoptosis/// sensory perception of sound///DNA repair///
nucleotide-excision repair///response to DNA damage stimulus///DNA
repair UNG uracil-DNA glycosylase carbohydrate metabolism///DNA
repair///base-excision repair///response to DNA damage
stimulus///DNA repair///DNA repair ERCC5 excision repair
cross-complementing transcription-coupled nucleotide-excision
repair/// rodent repair deficiency, nucleotide-excision
repair///sensory perception of sound/// complementation group 5
(xeroderma DNA repair///response to DNA damage stimulus///
pigmentosum, complementation group G nucleotide-excision repair
(Cockayne syndrome)) MLH1 mutL homolog 1, colon cancer, mismatch
repair///cell cycle///negative regulation of nonpolyposis type 2
(E. coli) progression through cell cycle///DNA repair/// mismatch
repair///response to DNA damage stimulus LIG1 ligase I, DNA,
ATP-dependent DNA replication///DNA repair///DNA recombination///
cell cycle///morphogenesis///cell division///DNA repair///response
to DNA damage stimulus///DNA metabolism NBN nibrin DNA damage
checkpoint///cell cycle checkpoint/// double-strand break repair
NBN nibrin DNA damage checkpoint///cell cycle checkpoint///
double-strand break repair NBN nibrin DNA damage checkpoint///cell
cycle checkpoint/// double-strand break repair MSH6 mutS homolog 6
(E. coli) mismatch repair///DNA metabolism///DNA repair/// mismatch
repair///response to DNA damage stimulus POLD4 polymerase
(DNA-directed), delta 4 DNA replication///DNA replication RFC5
replication factor C (activator 1) 5, DNA replication///DNA
repair///DNA replication 36.5 kDa RFC5 replication factor C
(activator 1) 5, DNA replication///DNA repair///DNA replication
36.5 kDa DDB2/// damage-specific DNA binding protein 2,
nucleotide-excision repair///regulation of transcription, LHX3 48
kDa///LIM homeobox 3 DNA-dependent///organ morphogenesis///DNA
repair/// response to DNA damage stimulus///DNA repair///
transcription///regulation of transcription POLD1 polymerase (DNA
directed), delta 1, DNA replication///DNA repair///response to
UV/// catalytic subunit 125 kDa DNA replication FANCG Fanconi
anemia, complementation cell cycle checkpoint///DNA repair///DNA
repair/// group G response to DNA damage stimulus///regulation of
progression through cell cycle POLB polymerase (DNA directed), beta
DNA-dependent DNA replication///DNA repair///DNA replication///DNA
repair///response to DNA damage stimulus XRCC1 X-ray repair
complementing defective single strand break repair repair in
Chinese hamster cells 1 MPG N-methylpurine-DNA glycosylase
base-excision repair///DNA dealkylation///DNA repair///
base-excision repair///response to DNA damage stimulus RFC2
replication factor C (activator 1) 2, DNA replication 40 kDa ERCC1
excision repair cross-complementing nucleotide-excision
repair///morphogenesis/// rodent repair deficiency,
nucleotide-excision repair///DNA repair///response to
complementation group 1 (includes DNA damage stimulus overlapping
antisense sequence) TDG thymine-DNA glycosylase carbohydrate
metabolism///base-excision repair///DNA repair///response to DNA
damage stimulus TDG thymine-DNA glycosylase carbohydrate
metabolism///base-excision repair///DNA repair///response to DNA
damage stimulus FANCA Fanconi anemia, complementation group DNA
repair///protein complex assembly///DNA repair/// A///Fanconi
anemia, complementation response to DNA damage stimulus group A
RFC4 replication factor C (activator 1) 4, DNA replication///DNA
strand elongation///DNA 37 kDa repair///phosphoinositide-mediated
signaling///DNA replication RFC3 replication factor C (activator 1)
3, DNA replication///DNA strand elongation 38 kDa RFC3 replication
factor C (activator 1) 3, DNA replication///DNA strand elongation
38 kDa APEX2 APEX nuclease (apurinic/apyrimidinic DNA
repair///response to DNA damage stimulus endonuclease) 2 RAD1 RAD1
homolog (S. pombe) DNA repair///cell cycle checkpoint///cell cycle
checkpoint///DNA damage checkpoint///DNA repair/// response to DNA
damage stimulus///meiotic prophase I RAD1 RAD1 homolog (S. pombe)
DNA repair///cell cycle checkpoint///cell cycle checkpoint///DNA
damage checkpoint///DNA repair/// response to DNA damage
stimulus///meiotic prophase I BRCA1 breast cancer 1, early onset
regulation of transcription from RNA polymerase II
promoter///regulation of transcription from RNA polymerase III
promoter///DNA damage response, signal transduction by p53 class
mediator resulting in transcription of p21 class mediator///cell
cycle/// protein ubiquitination///androgen receptor signaling
pathway///regulation of cell proliferation///regulation of
apoptosis///positive regulation of DNA repair/// negative
regulation of progression through cell cycle/// positive regulation
of transcription, DNA-dependent/// negative regulation of centriole
replication///DNA damage response, signal transduction resulting in
induction of apoptosis///DNA repair///response to DNA damage
stimulus///protein ubiquitination///DNA repair///regulation of DNA
repair///apoptosis/// response to DNA damage stimulus EXO1
exonuclease 1 DNA repair///DNA repair///mismatch repair///DNA
recombination FEN1 flap structure-specific endonuclease 1 DNA
replication///double-strand break repair///UV
protection///phosphoinositide-mediated signaling/// DNA
repair///DNA replication///DNA repair///DNA repair FEN1 flap
structure-specific endonuclease 1 DNA replication///double-strand
break repair///UV protection///phosphoinositide-mediated
signaling/// DNA repair///DNA replication///DNA repair///DNA repair
MLH3 mutL homolog 3 (E. coli) mismatch repair///meiotic
recombination///DNA repair/// mismatch repair///response to DNA
damage stimulus/// mismatch repair MGMT O-6-methylguanine-DNA DNA
ligation///DNA repair///response to DNA methyltransferase damage
stimulus RAD51 RAD51 homolog (RecA homolog, E. double-strand break
repair via homologous coli) (S. cerevisiae) recombination///DNA
unwinding during replication/// DNA repair///mitotic
recombination///meiosis/// meiotic recombination///positive
regulation of DNA ligation///protein homooligomerization///response
to DNA damage stimulus///DNA metabolism///DNA repair///response to
DNA damage stimulus///DNA repair///DNA recombination///meiotic
recombination/// double-strand break repair via homologous
recombination///DNA unwinding during replication RAD51 RAD51
homolog (RecA homolog, E. double-strand break repair via homologous
coli) (S. cerevisiae) recombination///DNA unwinding during
replication/// DNA repair///mitotic recombination///meiosis///
meiotic recombination///positive regulation of DNA
ligation///protein homooligomerization///response to DNA damage
stimulus///DNA metabolism///DNA repair///response to DNA damage
stimulus///DNA repair///DNA recombination///meiotic
recombination/// double-strand break repair via homologous
recombination///DNA unwinding during replication XRCC4 X-ray repair
complementing defective DNA repair///double-strand break
repair///DNA repair in Chinese hamster cells 4 recombination///DNA
recombination///response to DNA damage stimulus XRCC4 X-ray repair
complementing defective DNA repair///double-strand break
repair///DNA repair in Chinese hamster cells 4 recombination///DNA
recombination///response to DNA damage stimulus RECQL RecQ
protein-like (DNA helicase Q1- DNA repair///DNA metabolism like)
ERCC8 excision repair cross-complementing DNA
repair///transcription///regulation of rodent repair deficiency,
transcription, DNA-dependent///sensory perception of
complementation group 8 sound///transcription-coupled
nucleotide-excision repair FANCC Fanconi anemia, complementation
group DNA repair///DNA repair///protein complex assembly/// C
response to DNA damage stimulus OGG1 8-oxoguanine DNA glycosylase
carbohydrate metabolism///base-excision repair///DNA
repair///base-excision repair///response to DNA damage
stimulus///DNA repair MRE11A MRE11 meiotic recombination 11
regulation of mitotic recombination///double-strand homolog A (S.
cerevisiae) break repair via nonhomologous end-joining///
telomerase-dependent telomere maintenance///meiosis/// meiotic
recombination///DNA metabolism///DNA repair///double-strand break
repair///response to DNA damage stimulus///DNA
repair///double-strand break repair///DNA recombination RAD52 RAD52
homolog (S. cerevisiae) double-strand break repair///mitotic
recombination/// meiotic recombination///DNA repair///DNA
recombination///response to DNA damage stimulus WRN Werner syndrome
DNA metabolism///aging XPA xeroderma pigmentosum,
nucleotide-excision repair///DNA repair///response to
complementation group A DNA damage stimulus///DNA
repair///nucleotide- excision repair BLM Bloom syndrome DNA
replication///DNA repair///DNA recombination/// antimicrobial
humoral response (sensu Vertebrata)///
DNA metabolism///DNA replication OGG1 8-oxoguanine DNA glycosylase
carbohydrate metabolism///base-excision repair///DNA
repair///base-excision repair///response to DNA damage
stimulus///DNA repair MSH3 mutS homolog 3 (E. coli) mismatch
repair///DNA metabolism///DNA repair/// mismatch repair///response
to DNA damage stimulus POLE2 polymerase (DNA directed), epsilon 2
DNA replication///DNA repair///DNA replication (p59 subunit) RAD51C
RAD51 homolog C (S. cerevisiae) DNA repair///DNA
recombination///DNA metabolism/// DNA repair///DNA
recombination///response to DNA damage stimulus LIG4 ligase IV,
DNA, ATP-dependent single strand break repair///DNA
replication///DNA recombination///cell cycle///cell division///DNA
repair///response to DNA damage stimulus ERCC6 excision repair
cross-complementing DNA repair///transcription///regulation of
rodent repair deficiency, transcription,
DNA-dependent///transcription from RNA complementation group 6
polymerase II promoter///sensory perception of sound LIG3 ligase
III, DNA, ATP-dependent DNA replication///DNA repair///cell
cycle///meiotic recombination///spermatogenesis///cell division///
DNA repair///DNA recombination///response to DNA damage stimulus
RAD17 RAD17 homolog (S. pombe) DNA replication///DNA repair///cell
cycle///response to DNA damage stimulus XRCC2 X-ray repair
complementing defective DNA repair///DNA
recombination///meiosis///DNA repair in Chinese hamster cells 2
metabolism///DNA repair///response to DNA damage stimulus MUTYH
mutY homolog (E. coli) carbohydrate metabolism///base-excision
repair/// mismatch repair///cell cycle///negative regulation of
progression through cell cycle///DNA repair///response to DNA
damage stimulus///DNA repair RFC1 replication factor C (activator
1) 1, DNA-dependent DNA replication///transcription/// 145
kDa///replication factor C (activator regulation of transcription,
DNA-dependent/// 1) 1, 145 kDa telomerase-dependent telomere
maintenance///DNA replication///DNA repair RFC1 replication factor
C (activator 1) 1, DNA-dependent DNA replication///transcription///
145 kDa regulation of transcription, DNA-dependent///
telomerase-dependent telomere maintenance///DNA replication///DNA
repair BRCA2 breast cancer 2, early onset regulation of progression
through cell cycle///double- strand break repair via homologous
recombination/// DNA repair///establishment and/or maintenance of
chromatin architecture///chromatin remodeling/// regulation of S
phase of mitotic cell cycle///mitotic checkpoint///regulation of
transcription///response to DNA damage stimulus RAD50 RAD50 homolog
(S. cerevisiae) regulation of mitotic recombination///double-strand
break repair///telomerase-dependent telomere maintenance///cell
cycle///meiosis///meiotic recombination///chromosome organization
and biogenesis///telomere maintenance///DNA repair/// response to
DNA damage stimulus///DNA repair/// DNA recombination DDB1
damage-specific DNA binding protein 1, nucleotide-excision
repair///ubiquitin cycle///DNA 127 kDa repair///response to DNA
damage stimulus///DNA repair XRCC5 X-ray repair complementing
defective double-strand break repair via nonhomologous end- repair
in Chinese hamster cells 5 joining///DNA recombination///DNA
repair///DNA (double-strand-break rejoining; Ku
recombination///response to DNA damage stimulus/// autoantigen, 80
kDa) double-strand break repair XRCC5 X-ray repair complementing
defective double-strand break repair via nonhomologous end- repair
in Chinese hamster cells 5 joining///DNA recombination///DNA
repair///DNA (double-strand-break rejoining; Ku
recombination///response to DNA damage stimulus/// autoantigen, 80
kDa) double-strand break repair PARP1 poly (ADP-ribose) polymerase
family, DNA repair///transcription from RNA polymerase II member 1
promoter///protein amino acid ADP-ribosylation/// DNA
metabolism///DNA repair///protein amino acid
ADP-ribosylation///response to DNA damage stimulus POLE3 polymerase
(DNA directed), epsilon 3 DNA replication (p17 subunit) RFC1
replication factor C (activator 1) 1, DNA-dependent DNA
replication///transcription/// 145 kDa regulation of transcription,
DNA-dependent/// telomerase-dependent telomere maintenance///DNA
replication///DNA repair RAD50 RAD50 homolog (S. cerevisiae)
regulation of mitotic recombination///double-strand break
repair///telomerase-dependent telomere maintenance///cell
cycle///meiosis///meiotic recombination///chromosome organization
and biogenesis///telomere maintenance///DNA repair/// response to
DNA damage stimulus///DNA repair/// DNA recombination XPC xeroderma
pigmentosum, nucleotide-excision repair///DNA repair///nucleotide-
complementation group C excision repair///response to DNA damage
stimulus/// DNA repair MSH2 mutS homolog 2, colon cancer, mismatch
repair///postreplication repair///cell cycle/// nonpolyposis type 1
(E. coli) negative regulation of progression through cell cycle///
DNA metabolism///DNA repair///mismatch repair/// response to DNA
damage stimulus///DNA repair RPA3 replication protein A3, 14 kDa
DNA replication///DNA repair///DNA replication MBD4 methyl-CpG
binding domain protein 4 base-excision repair///DNA
repair///response to DNA damage stimulus///DNA repair MBD4
methyl-CpG binding domain protein 4 base-excision repair///DNA
repair///response to DNA damage stimulus///DNA repair NTHL1 nth
endonuclease III-like 1 (E. coli) carbohydrate
metabolism///base-excision repair/// nucleotide-excision repair,
DNA incision, 5'-to lesion/// DNA repair///response to DNA damage
stimulus PMS2/// PMS2 postmeiotic segregation increased mismatch
repair///cell cycle///negative regulation of PMS2CL 2 (S.
cerevisiae)///PMS2-C terminal-like progression through cell
cycle///DNA repair/// mismatch repair///response to DNA damage
stimulus/// mismatch repair RAD51C RAD51 homolog C (S. cerevisiae)
DNA repair///DNA recombination///DNA metabolism/// DNA repair///DNA
recombination///response to DNA damage stimulus UNG2 uracil-DNA
glycosylase 2 regulation of progression through cell cycle///
carbohydrate metabolism///base-excision repair///DNA
repair///response to DNA damage stimulus APEX1 APEX nuclease
(multifunctional DNA base-excision repair///transcription from RNA
repair enzyme) 1 polymerase II promoter///regulation of DNA
binding/// DNA repair///response to DNA damage stimulus ERCC4
excision repair cross-complementing nucleotide-excision
repair///nucleotide-excision repair/// rodent repair deficiency,
DNA metabolism///DNA repair///response to DNA complementation group
4 damage stimulus RAD1 RAD1 homolog (S. pombe) DNA repair///cell
cycle checkpoint///cell cycle checkpoint///DNA damage
checkpoint///DNA repair/// response to DNA damage
stimulus///meiotic prophase I RECQL5 RecQ protein-like 5 DNA
repair///DNA metabolism///DNA metabolism MSH5 mutS homolog 5 (E.
coli) DNA metabolism///mismatch repair///mismatch repair///
meiosis///meiotic recombination///meiotic prophase II///meiosis
RECQL RecQ protein-like (DNA helicase Q1- DNA repair///DNA
metabolism like) RAD52 RAD52 homolog (S. cerevisiae) double-strand
break repair///mitotic recombination/// meiotic recombination///DNA
repair///DNA recombination///response to DNA damage stimulus XRCC4
X-ray repair complementing defective DNA repair///double-strand
break repair///DNA repair in Chinese hamster cells 4
recombination///DNA recombination///response to DNA damage stimulus
XRCC4 X-ray repair complementing defective DNA
repair///double-strand break repair///DNA repair in Chinese hamster
cells 4 recombination///DNA recombination///response to DNA damage
stimulus RAD17 RAD17 homolog (S. pombe) DNA replication///DNA
repair///cell cycle///response to DNA damage stimulus MSH3 mutS
homolog 3 (E. coli) mismatch repair///DNA metabolism///DNA
repair/// mismatch repair///response to DNA damage stimulus MRE11A
MRE11 meiotic recombination 11 regulation of mitotic
recombination///double-strand homolog A (S. cerevisiae) break
repair via nonhomologous end-joining/// telomerase-dependent
telomere maintenance///meiosis/// meiotic recombination///DNA
metabolism///DNA repair///double-strand break repair///response to
DNA damage stimulus///DNA repair///double-strand break repair///DNA
recombination MSH6 mutS homolog 6 (E. coli) mismatch repair///DNA
metabolism///DNA repair/// mismatch repair///response to DNA damage
stimulus MSH6 mutS homolog 6 (E. coli) mismatch repair///DNA
metabolism///DNA repair/// mismatch repair///response to DNA damage
stimulus RECQL5 RecQ protein-like 5 DNA repair///DNA
metabolism///DNA metabolism BRCA1 breast cancer 1, early onset
regulation of transcription from RNA polymerase II
promoter///regulation of transcription from RNA polymerase III
promoter///DNA damage response, signal transduction by p53 class
mediator resulting in transcription of p21 class mediator///cell
cycle/// protein ubiquitination///androgen receptor signaling
pathway///regulation of cell proliferation///regulation of
apoptosis///positive regulation of DNA repair/// negative
regulation of progression through cell cycle/// positive regulation
of transcription, DNA-dependent/// negative regulation of centriole
replication///DNA damage response, signal transduction resulting in
induction of apoptosis///DNA repair///response to DNA damage
stimulus///protein ubiquitination///DNA repair///regulation of DNA
repair///apoptosis/// response to DNA damage stimulus RAD52 RAD52
homolog (S. cerevisiae) double-strand break repair///mitotic
recombination/// meiotic recombination///DNA repair///DNA
recombination///response to DNA damage stimulus POLD3 polymerase
(DNA-directed), delta 3, DNA synthesis during DNA repair///mismatch
repair/// accessory subunit DNA replication MSH5 mutS homolog 5 (E.
coli) DNA metabolism///mismatch repair///mismatch repair///
meiosis///meiotic recombination///meiotic prophase II///meiosis
ERCC2 excision repair cross-complementing transcription-coupled
nucleotide-excision repair/// rodent repair deficiency,
transcription///regulation of transcription, DNA- complementation
group 2 (xeroderma dependent///transcription from RNA polymerase II
pigmentosum D) promoter///induction of apoptosis///sensory
perception of sound///nucleobase, nucleoside, nucleotide and
nucleic acid metabolism///nucleotide-excision repair RECQL4 RecQ
protein-like 4 DNA repair///development///DNA metabolism PMS1 PMS1
postmeiotic segregation increased mismatch repair///regulation of
transcription, DNA- 1 (S. cerevisiae) dependent///cell
cycle///negative regulation of progression through cell
cycle///mismatch repair/// DNA repair///response to DNA damage
stimulus ZFP276 zinc finger protein 276 homolog (mouse)
transcription///regulation of transcription, DNA- dependent MBD4
methyl-CpG binding domain protein 4 base-excision repair///DNA
repair///response to DNA damage stimulus///DNA repair MBD4
methyl-CpG binding domain protein 4 base-excision repair///DNA
repair///response to DNA damage stimulus///DNA repair
MLH3 mutL homolog 3 (E. coli) mismatch repair///meiotic
recombination///DNA repair/// mismatch repair///response to DNA
damage stimulus/// mismatch repair FANCA Fanconi anemia,
complementation group DNA repair///protein complex assembly///DNA
repair/// A response to DNA damage stimulus POLE polymerase (DNA
directed), epsilon DNA replication///DNA repair///DNA
replication/// response to DNA damage stimulus XRCC3 X-ray repair
complementing defective DNA repair///DNA recombination///DNA
metabolism/// repair in Chinese hamster cells 3 DNA repair///DNA
recombination///response to DNA damage stimulus///response to DNA
damage stimulus MLH3 mutL homolog 3 (E. coli) mismatch
repair///meiotic recombination///DNA repair/// mismatch
repair///response to DNA damage stimulus/// mismatch repair NBN
nibrin DNA damage checkpoint///cell cycle checkpoint///
double-strand break repair SMUG1 single-strand selective
monofunctional carbohydrate metabolism///DNA repair///response to
uracil DNA glycosylase DNA damage stimulus FANCF Fanconi anemia,
complementation group DNA repair///response to DNA damage stimulus
F NEIL1 nei endonuclease VIII-like 1 (E. coli) carbohydrate
metabolism///DNA repair///response to DNA damage stimulus FANCE
Fanconi anemia, complementation group DNA repair///response to DNA
damage stimulus E MSH5 mutS homolog 5 (E. coli) DNA
metabolism///mismatch repair///mismatch repair/// meiosis///meiotic
recombination///meiotic prophase II///meiosis RECQL5 RecQ
protein-like 5 DNA repair///DNA metabolism///DNA metabolism
[0033] Therefore, it should be appreciated that any one or more of
the above genes in Tables 1-3 can be assessed for mutations (which
may be further classified or assessed into mutations affecting
function or silent mutations), for copy number, and/or for
expression strength, as well as RNA splice variants and differences
in polyadenylation or other parameters that affect stability or
half-life of a transcript. Likewise, protein quantity and/or
protein activities for the corresponding proteins encoded by the
genes of Tables 1-3 may be determined using mass spec or in vitro
assays well known in the art. Consequently, the repair status of a
cell can be assessed using the omics data across a wide variety of
repair mechanisms. As such, one or more deficiencies (functional
and/or by decreased quantity) in DNA repair genes relative to
normal may be indicative of a diseased cell or lack of repair
capability, which in turn may be indicative for treatment success
using DNA damaging agents. On the other hand, over-activity or
overexpression (relative to a healthy cell of the same individual)
of one or more DNA repair genes may be indicative of DNA damage,
presence or exposure to a DNA damaging environment or agent.
Moreover, functional defects in DNA repair genes may be indicative
of a predisposition to hypermutations.
[0034] As will be also readily appreciated, the DNA repair function
as assessed by omics data can be correlated with damage patterns
that are present or that can be expected. Thus, analysis of
mutation signatures (see e.g.,
URL:cancer.sanger.ac.uk/cosmic/signatures) in conjunction with the
teachings presented herein is also contemplated. For example,
mutation signatures 2 and 13 have been attributed to activity of
the AID/APOBEC family of cytidine deaminases, while signature 4
exhibits transcriptional strand bias for C>A mutations, which is
compatible with the notion that damage to guanine is repaired by
transcription-coupled nucleotide excision repair. Mutation
signature 26 is associated with defective DNA mismatch repair. Most
typically, the observed or expected mutation signatures will
generally correlate with a reduced or increased activity of DNA
corresponding repair genes, the type of tumor, and/or exposure to
DNA damaging agents (environmental, or drug-associated).
[0035] Of course, it should be appreciated that analyses presented
herein may be performed over specific and diverse populations to
thereby obtain reference values for the specific populations, such
as across various health associated states (e.g., healthy,
diagnosed with a specific disease and/or disease state, which may
or may not be inherited, or which may or may not be associated with
impaired DNA repair), a specific age or age bracket, a specific
ethnic group that may or may not be associated with longevity or
high morbidity/mortality (e.g., Okinawa Japanese, Nepalese, Sri
Lankans, etc.), and/or pharmaceutical treatment (e.g., treatment
with DNA alkylating agents, DNA crosslinkers, DNA intercalators, or
platinum adducts). Of course, populations may also be enlisted from
databases with known omics information, and especially publically
available omics information from cancer patients (e.g., TCGA,
COSMIC, etc.) and proprietary databases from a large variety of
individuals that may be healthy or diagnosed with a disease.
Likewise, it should be appreciated that the population records may
also be indexed over time for the same individual or group of
individuals, which advantageously allows detection of shifts or
changes in the genes and pathways associated with RNA repair.
[0036] Thus, it should be recognized that contemplated systems and
methods allow for a large cross sectional database for DNA repair
gene activity, which in turn allows the generation of a risk matrix
that may be based on individual DNA repair gene scores, on ratio
scores, sum scores, differential scores, etc. In particularly
preferred aspects, it is contemplated that an error score can be
established for one or more DNA repair genes, and that the score
may be reflective of or even prognostic for various diseases that
are at least in part due to mutations in DNA repair genes and/or
pathways. For example, especially suitable error scores may involve
scores for one or more genes associated with one or more types of
DNA repair (e.g., base excision repair, homologous recombination
repair, etc.) relative to another gene that may or may not be
associated with one type of DNA repair (e.g., TP53, Fas, bcl-2,
CHK2, Non-homologous end-joining repair gene, etc.). In another
example, contemplated error scores may involve scores for one or
more genes associated with one or more types of DNA repair (e.g.,
base excision repair, homologous recombination repair, etc.)
relative to an overall mutation rate to so better identify DNA
repair relevant mutations over `background` mutations. In still
other examples, mutations in some DNA repair genes may be `leading
indicators` or triggers to activate other DNA repair mechanism such
as p53 mediated repair. Identification of such triggers may
advantageously allow for early diagnosis of repair events, or may
be used to trigger repair events.
[0037] Based on the particular quantitation and/or analysis of the
omics data, it should be noted that various calculations can be
performed. For example, the omics data may be used to generate a
general error status for an individual (or tumor within an
individual), or to associate the number and/or type of alterations
in DNA repair genes to identify a `tipping point` for one or more
DNA repair gene mutations after which a general mutation rate
skyrockets. For example, where a rate or number of mutations in
ERCC1 and other DNA repair genes could have only minor systemic
consequence, addition of further mutations to TP53 may result in a
catastrophic increase in mutation rates. Thus, and viewed from a
different perspective, mutations in the genes associated with DNA
may be used to estimate the risk of occurrence for a DNA
damage-based disease, and especially cancer and age-related
diseases. In still further contemplated uses, so obtained omics
information may be analyzed in one or more pathway analysis
algorithms (e.g., PARADIGM) to so identify affected pathways and to
so possibly adjust treatment where treatment employs DNA damaging
agents. Pathway analysis algorithms may also be used to in silico
modulate expression of one or more DNA repair genes, which may
results in desirable or even unexpected in silico treatment
outcomes, which may be translated into the clinic. Likewise,
various machine learning algorithms may be employed to associate a
disease parameter (e.g., type of disease, stage of disease,
treatability of a disease with specific drug) with the omics data
for the genes associated with DNA repair) to so identify a specific
mutation pattern as being correlated with a particular condition or
drug sensitivity.
[0038] In still further contemplated aspects, it should be
appreciated that once one or more genes associated with DNA repair
have been identified as dysfunctional (e.g., over-expressed,
under-expressed, mutated, truncated, splice variant present, etc.),
drugs can be identified to counteract the dysfunctional gene. As
noted above, such drugs can be identified using large small
molecule libraries, computational approaches, and/or data from the
public domain. Moreover, in silico simulations using pathway models
may be employed to identify such drugs. Consequently, it should be
appreciated that contemplated system and methods may not only be of
diagnostic value, but also be employed to identify and use drugs
that counteract mutation-related diseases, and especially cancer
and age-related diseases. In such systems, one or more drugs can
then be administered to an individual to counteract DNA repair
activity, and/or to treat a specific cell population that is
characterized by a DNA repair signature.
[0039] Therefore, contemplated omics analyses are also particularly
useful for monitoring treatment of a patient that is subject to a
pharmaceutical intervention. Such monitoring will advantageously
include detection and/or quantification of diseased cells having a
specific repair signature, detection of triggering DNA repair in
healthy tissue during treatment with DNA damaging agents, detection
of development of treatment resistant clonal populations having a
specific repair signature, and detection of disease recurrence
where the diseased cells have a particular repair signature. Viewed
from a different perspective, the signatures may also be used to
identify whether or not a cell population is likely sensitive to
treatment with DNA damaging agents. Similarly, the signatures may
also be used in a combination treatment where an individual
receives treatment with a DNA damaging agent and at the same time
one or more pharmaceutical agents that inhibit the corresponding
DNA repair genes required to repair the damage brought on by the
DNA damaging agent. Such strategy may be readily monitored using
contemplated omics tests. Thus, and viewed from yet another
perspective, contemplated methods may be employed to specifically
identify and then target DNA repair mechanisms (e.g., using PARP
inhibitors, Chk1-2 inhibitors, WEE-1 inhibitors, or ATR inhibitors)
that may be used by a cell to counteract treatment with a DNA
damaging agent.
EXAMPLE 1
[0040] A whole blood sample is provided and divided into two
aliquots. A first aliquot is used to isolate cell free RNA, cfRNA
(and where desired cell free DNA, cfDNA) as described below.
However, various other bodily fluids are also deemed appropriate so
long as cfRNA is present in such fluids. Appropriate fluids include
saliva, ascites fluid, spinal fluid, urine, etc, which may be
fresh, chemically preserved, or refrigerated or frozen. For
example, specimens can be accepted as 10 ml of whole blood drawn
into commercially available cell-free RNA BCT.RTM. tubes or
cell-free DNA BCT.RTM. tubes (Streck, 7002 S. 109 St., Omaha, Nebr.
68128) containing RNA or DNA stabilizers, respectively.
Advantageously, cfRNA is stable in whole blood in the cell-free RNA
BCT tubes for seven days while cfDNA is stable in whole blood in
the cell-free DNA BCT Tubes for fourteen days, allowing time for
shipping of patient samples from world-wide locations without the
degradation of cfRNA or cfDNA. Moreover, it is generally preferred
that the cfRNA is isolated using RNA stabilization agents that will
not or substantially not (e.g., equal or less than 1%, or equal or
less than 0.1%, or equal or less than 0.01%, or equal or less than
0.001%) lyse blood cells. Viewed from a different perspective, RNA
stabilization reagents will not lead to a substantial increase
(e.g., increase in total RNA no more than 10%, or no more than 5%,
or no more than 2%, or no more than 1%) in RNA quantities in serum
or plasma after the reagents are combined with blood.
[0041] Most typically, but not necessarily, the first aliquot is
centrifuged in the presence of an RNase inhibitor, a preservative
agent, a metabolic inhibitor, and a chelator. Moreover, it is
generally preferred that the step of centrifuging whole blood is
performed under conditions that preserve the integrity of cellular
components. For example, the first RCF may be between 700 and 2,500
(e.g., 1,600), and/or the second RCF may be between 7,000 and
25,000 (e.g., 16,000), wherein centrifugation at the first RCF is
performed between 15-25 minutes (e.g., 20 minutes) and wherein the
centrifugation at the second RCF is performed between 5-15 minutes
(e.g., 10 minutes). Where desired or required, cfRNA may be stored
at -80.degree. C. and/or cDNA prepared from the cfRNA may be stored
at -4.degree. C.
[0042] A second aliquot of the whole blood sample can be
centrifuged in an evacuated blood collection tube to separate the
cells from the serum/plasma. Once isolated, the cells can be washed
in isotonic ringer solution and then lysed to so prepare DNA and
RNA using one or more commercially available test kits (e.g.,
Qiagen DNA blood mini kit, Qiagen RNA blood mini kit).
[0043] For both analyses, DNA and RNA sequencing is performed. In
addition, quantitative RNA analysis is employed to obtain
transcriptomics information. Where available, proteomics analysis
is performed using selected reaction monitoring for at least two,
or at least 4, or at least 10, or at least 20 different proteins
associated with DNA repair. So obtained omics information can then
be processed using pathway analysis (especially using PARADIGM) to
identify any impact of any mutations on DNA repair pathways.
EXAMPLE 2
[0044] A whole blood sample is drawn from a patient diagnosed with
cancer and processed as noted in Example 1 above. In addition, a
fresh tumor biopsy is obtained and a full omics analysis performed
in which DNA sequencing is whole genome sequencing at a depth of at
least 20.times. for DNA and RNA. In addition, quantitative RNA
analysis is employed to obtain transcriptomics information. Where
available, proteomics analysis is performed using selected reaction
monitoring for at least two, or at least 4, or at least 10, or at
least 20 different proteins associated with DNA repair. Where
desired, proteomics analysis is performed using selected reaction
monitoring for at least two, or at least 4, or at least 10, or at
least 20 different proteins associated with DNA repair. So obtained
omics information can then be processed using pathway analysis
(especially using PARADIGM) to identify any impact of any mutations
on DNA repair pathways.
EXAMPLE 3
[0045] Once omics analysis for a patient sample (e.g., of Example
2) is concluded, changes in DNA, RNA, and protein (activities)
relative to omics data of age-matched healthy individuals are
noted. Such changes may be labeled idiosyncratic where no
statistical association with a known disease pattern is observed,
or changes may be associated with a pattern that is characteristic
of a disease. As noted above, analysis may include observation on
individual genes associated with DNA repair, or on multiple genes,
alone or in various relationships (e.g., ratio, sum, etc.).
EXAMPLE 4
[0046] A tumor biopsy and a biopsy of corresponding non-tumor
tissue (or a blood sample) is obtained from an individual. The
tumor biopsy is then subjected to DNA sequencing and RNAseq with
quantification of expressed RNA in the tumor cells. Mutational
status for all DNA repair genes is determined as well as the
transcription strength, for both the biopsy sample and the
corresponding non-tumor tissue. Differences in repair status are
ascertained and treatment with DNA damaging agents (e.g., using
crosslinkers, intercalating agents, etc.) is started. Treatment is
then monitored either by re-biopsy of the tumor or by isolation and
analysis of cfDNA and cfRNA for DNA repair genes as discussed
above. Where an increase in DNA repair gene expression is noted in
the tumor sample, inhibitors for DNA repair may be administered.
Moreover, pathway analysis (e.g., using PARADIGM) can be performed
using the omics data to identify further treatment options that
will selectively interfere with tumor DNA repair. During such
follow-up, repair signatures may be obtained for the tumor to
identify clonal development, evolution of resistance, and or tumor
status. Upon conclusion, repair signatures may be obtained
(typically from cell free DNA and cell free RNA to detect tumor
specific repair signatures, which may be indicative of
recurrence.
[0047] In some embodiments, the numbers expressing quantities of
ingredients, properties such as concentration, reaction conditions,
and so forth, used to describe and claim certain embodiments of the
invention are to be understood as being modified in some instances
by the term "about." Accordingly, in some embodiments, the
numerical parameters set forth in the written description and
attached claims are approximations that can vary depending upon the
desired properties sought to be obtained by a particular
embodiment. In some embodiments, the numerical parameters should be
construed in light of the number of reported significant digits and
by applying ordinary rounding techniques. Notwithstanding that the
numerical ranges and parameters setting forth the broad scope of
some embodiments of the invention are approximations, the numerical
values set forth in the specific examples are reported as precisely
as practicable. The numerical values presented in some embodiments
of the invention may contain certain errors necessarily resulting
from the standard deviation found in their respective testing
measurements. Unless the context dictates the contrary, all ranges
set forth herein should be interpreted as being inclusive of their
endpoints and open-ended ranges should be interpreted to include
only commercially practical values. Similarly, all lists of values
should be considered as inclusive of intermediate values unless the
context indicates the contrary.
[0048] As used in the description herein and throughout the claims
that follow, the meaning of "a," "an," and "the" includes plural
reference unless the context clearly dictates otherwise. Also, as
used in the description herein, the meaning of "in" includes "in"
and "on" unless the context clearly dictates otherwise.
[0049] It should be apparent to those skilled in the art that many
more modifications besides those already described are possible
without departing from the inventive concepts herein. The inventive
subject matter, therefore, is not to be restricted except in the
spirit of the appended claims. Moreover, in interpreting both the
specification and the claims, all terms should be interpreted in
the broadest possible manner consistent with the context. In
particular, the terms "comprises" and "comprising" should be
interpreted as referring to elements, components, or steps in a
non-exclusive manner, indicating that the referenced elements,
components, or steps may be present, or utilized, or combined with
other elements, components, or steps that are not expressly
referenced. Where the specification claims refers to at least one
of something selected from the group consisting of A, B, C . . .
and N, the text should be interpreted as requiring only one element
from the group, not A plus N, or B plus N, etc.
* * * * *